AUTOMATED RARE CELLS CAPTURE & LABELLING ESS TM Platform for the automation of a complex biological process

Transcription

1 AUTOMATED RARE CELLS CAPTURE & LABELLING ESS TM Platform for the automation of a complex biological process DATA PAGE Introduction 2 Method for CTCs capture and detection 3 Automation of the CTCs capture & detection with ESS TM 4 Conclusion 9 FOR ANY FURTHER INQUIRY, PLEASE CONTACT US AT: FLUIGENT SA CONTACT@FLUIGENT.COM

2 INTRODUCTION This application note describes the use of Fluigent microluidic luid handling solutions for the complete automation of a complex microluidic set-up. Because CTCs detection and labelling need a very precise and smooth low control, Fluigent MFCS TM associated with the Flow-Rate Control Module and the ESS TM platform, was the designated choice to be able to eiciently control the lows of all the solutions and samples. Circulating Tumor Cells (CTCs) are tumoral cells issued from a primary tumor. They transiently circulate in the blood stream until they stop in speciic organs to seed metastases. Metastases are involved in most cancer deaths and often develop characteristics which make them resistant to treatments that were eicient on the primary tumors. In this context, being able to study these CTCs represents several assets such as to monitor over time the disease progression as well as to develop the best-suited therapeutic strategies. Several methods have been studied to capture and detect CTCs. They usually include a large number of steps (injection of several diferent samples, bufers, etc) and can last several hours. This application note describes the automation of a 5-hour CTC capture and detection method including the precise volume control of more than 10 diferent solutions. MATERIAL USED TO CONTROL FLOWS PRODUCTS NAME & USE MFCS TM -EX Microluidic pressure pump to generate fast and pulseless fluid movement (345 mbar range) FLOWELL + Flow-Rate Control Module To control and monitor the low-rate on up to 8 channels FLUIWELL (1C & 4C) Reservoirs (2ml and 15ml) for cell samples and bufers ESS TM (Easy Switch Solution TM ) 2-SWITCH TM, M-SWITCH TM & SWITCHBOARD Fluid handling platform to automate any selection or injection of liquids P.2

3 METHOD FOR CTC CAPTURE & LABELLING Circulating tumor cells (CTCs) are cells from a primary tumor that circulate in the bloodstream (Figure 1). Thus, capturing, counting and characterizing (at both proteomic and genetic levels) CTCs, can give precious information about the development stage of the cancer, its nature, and consequently about the best-suited drug treatment. Analysing blood from a cancer patient for CTCs thus provides a liquid biopsy to the physician. Nonetheless, developing an instrument to perform the capture and the labelling of CTCs is highly challenging since CTCs are considered as rare cells: only one CTC for several millions of white blood cells lows in the blood. To achieve a suicient sensitivity and sensibility in the instrument, Institut Curie has developed the EPHESIA microluidic device (Figure 2). This microluidic chip is made of PDMS: a tree-like channel structure lows the luids in and out two 3cm-long chambers with a magnetic dot pattern integrated in the bottom layer of the chambers. OOD VESSEL Figure 1: CTCs pathway, Capture and Analysis of Circulating Tumor Cells in Microfluidics: From Conception to Clinical Validation, Julien Autebert, Physico-Chimie Curie, UMR168, 2013 P.3

4 THE PROCESS INCLUDES THE FOLLOWING STEPS:. 1. After surface treatment, magnetic beads, coated with EpCAM antibodies (antibodies complementary to the target biomarkers of the CTCs), are injected in the chambers. EpCAM is a surface antigen speciic to epithelial cells, a category of cells CTCs belong to. As no epithelial cells are present in the blood stream of a healthy person, all epithelial cells that are found in a blood sample can be identiied as CTCs.. 2. The magnetic ield is turned on. This magnetic ield is perpendicular to the chip bottom layer: under its inluence the magnetic beads self-assemble into columns that are spontaneously anchored on the magnetic patterns. The result is a sieve made of magnetic beads columns.. 3. After magnetic beads self-assembly, the sample containing white blood cells and CTCs is lowed through the columns. Due to the anti-epcam antibodies coating of the beads, only the CTCs speciically bind to the columns, while the rest of the sample exits the chip..4. Several washing steps and luorescent dyes injections are then done on the captured cells (Hoechst for DNA in the nucleus to check for nucleated cells, CD45 to discriminate leucocytes, Cytokeratin CK to identify epithelial cells, with FIX & PERM TM treatment). The whole process can take up to 5 hours and requires numerous steps with precise low-rate and volume control on the diferent injected solutions: it was thus very important to use a high performance low control solution capable of automating the whole set-up, controlling precisely the low-rates and the volumes injected while providing pulseless and responsive lows. 3mm Figure 2: Diagram and photography of the CTC capture chip, Design, modeling and characterization of microfluidic architectures for high fow rate, small footprint microluidic systems, Saias and al., copyright 2011, Lab on chip 3cm PDMS Sample 50µm Flow PDMS Magnetic Beads Anchorage P.4

5 / / /.- APPLICATION NOTE A U T abcti O N O F C T C s CAPTURE & LABELLING WITH ESS TM The ESS TM platform associated with the MFCS TM, the FRP and the Flow-Rate Control Module, provides key features for this application and its complex set-up (igure 3 and igure 4): The full automation of the set-up 1, saving time by providing a reliable control on the experiment without the need for the operator to stay close to the experiment. Low internal volume ( 12µl) reducing your samples and reagents consumption, so thus reducing the overall cost of your experiment Real time monitoring of the low rates, the pressures, the volumes and the valves positions, to follow each step of your protocol and adjust it quickly if needed. Fast and highly stable control of the pressures and the low-rates Figure 3: Photograph of the automated set-up for CTCs capture and detection FLUIWELL 4C # FLUIWELL 1C. 3. M-SWITCH TM #A. 4. SWITCHBOARD. 5 #1. to. 5 #5. 2-SWITCH TM #1 to #5. 6. CTC Chip #1.. 5 #2.. 5 # # MFCS TM. 1. CDEFGH IEFJKJEL MLDGKF NOKDGK 8!"#$%&TM '9 PQRwSTUtVWQUtfRrm XYZ[\]^_ ` >?@" AB#$!"#$%&TM '(!"#$%&TM #5 %$% )*+, TM TM TM Figure 4: Fluidic diagram of the automated set-up for CTCs capture and detection PBS Water Bleach Hoechst Fix CD45 Perm CK Water Beads Cells :;<= (1) Through scripts you can design to precisely meet your needs P.5

6 Az{ }~{ ~ ƒƒ ~ {z ƒ~ ƒƒˆ Š efghi j k lmnn lopqrsm pstlmuu Arqtvoqwtx To automate the whole set-up, two phases were necessary:.1. Automate the path selection inside the chip itself to inject beads, cells (samples) and bufer for the cell capture process.2. Provide an easy and automated way to bring to the chip all the diferent bufers and reagents (9 diferent liquids) needed for the biological process without adding inlets on the chip. There are 3 inlets and 1 outlet on this chip: depending on which inlet or outlet is closed, diferent paths can be taken to perform the diferent luidic steps. The automated path selection inside the chip was achieved by connecting a 2-SWITCH TM valve to each inlet/outlet, with one of their ports plugged. They thus act as luidic on/ of switches : one position lets the luid low through the 2-SWITCH TM whereas the other position closes the path (Figure 5 and 6). Figure 5: Photograph of the automated set-up for CTCs capture chip with its path selection 2-SWITCH TM valves. y. SWITCHBOARD.d#1. to.d#5. 2-SWITCH TM #1 to #5.6. CTC Chip.4..d#4..7..d# MFCS TM.6..d#3 2-SWITCH TM #5 Flow-Rate Platform FLOW UNIT S FLOW-UNIT Closed position Inlets Outlet Figure 6: Diagram of the automated set-up for CTCs capture chip and its path selection systems including 2-SWITCH TM valves 2-SWITCH TM #4 2-SWITCH TM #3 Cells Beads MFCS CTC CHIP 2-SWITCH TM #1 To M-SWITCH & buffers P.6

7 Thanks to these irst automation phases, illing processes are launched. They need to be done prior to the analysis to avoid any bubbles and save time during the experiment. The chip illing is done with a bufer handled by the M-SWITCH TM. Then, we proceed to the bead inlet illing and the cells (sample) inlet illing. Once illings are done, the analysis steps are then automated as well: beads are injected to form the magnetic beads columns that sieve the CTCs (Figure 7) when the magnetic ield is turned on. Then cells (sample) are smoothly injected: the CTCs speciically bind to the columns while the other cells low towards the outlet (Figure 8). Œ Ž Figure 7: Photograph of a part of one chip chamber with automatically formed magnetic beads columns Magnetic beads columns Tree-Like Channels Figure 8: Photograph of a part of one chip chamber with speciically captured cells on magnetic beads columns. Capture and Analysis of Circulating Tumor Cells in Microfluidics: From Conception to Clinical Validation, Julien Autebert, Physico-Chimie Curie, UMR168, 2013 Speciically captured cells Magnetic beads columns P.7

8 PHASE 2 : CELL LABELLING PROCESS BUFFER SELECTION SYSTEM As mentioned above, the second automation step consists in injecting 8 diferent bufers and reagents without adding inlets to the chip: all 8 liquids have to be injected inside the same microchannel. To do so, the solution consists in connecting all bufers and reagents to one M-SWITCH TM (which can be connected to up to 10 diferent solutions). The M-SWITCH TM links the central port to one of the external ports where the bufers are connected. We also use two 2-SWITCH TM (#1 and #2) to build a secondary path towards water and waste reservoirs (Figure 9). This way, bufers from the M-SWITCH TM #A can either be directed to the chip for cell labelling or to the secondary path (Figure 10 & 11) for illing and rinsing. Once these actuators put in place, the operator can write scripts to perform automatically all the luidic labelling steps plus illing and washing of all branches of the circuit. Figure 9: Photograph of the buffer selection system with one M-SWITCH TM for bufer connection and selection and two 2-SWITCH TM for main or secondary luidic path selection Water.4. š Water œ ž.3. M-SWITCH TM #A.4. SWITCHBOARD TM. #1. to. #5. 2-SWITCH TM #1 to #5. 3. Bleach. Hoechst. Fix. Cd45.. (common). #2.. #1. Perm CK œÿ ž Closed position Main path selection Secondary path selection CTC chip M-SWITCH TM #A 2-SWITCH TM #1 Figure 10: Diagram of the buffer selection system with one M-SWITCH TM for bufer connection and selection and two 2-SWITCH TM for main or secondary luidic path selection PBS Water Bleach Hoechst Fix CK Perm Cd45 Water 2-SWITCH TM #2 MFCS P.8

9 CONCLUSION During all the experiment, luids were handled by 2 MFCS TM pressure controller through the Flow-rate Control Module 1 providing the full control of the diferent low rates while beneiting from the pressure actuation: stability/pulseless low (standard deviation <0.1% 2 ) and responsiveness (settling time below 200ms 2 ) These beneits were especially important at all stages for this application in order to: Automate the 39 steps included in this speciic application from illing to cells labelling and all washing steps Keep the integrity of the columns and the cells bound to the columns while all bufers and reagents low inside the chip thanks to the pulseless lows provided by the MFCS TM and the Flow-Rate Control Module 1, especially by setting a maximum pressure to reach and thus avoiding any chip damages Quickly switch from a liquid to another thanks to the responsiveness of the ESS TM and the MFCS TM -Flow-Rate Control Module Reduce bufer and reagent consumptions, thus their costs, thanks to the low internal volume ( 12µl) of the ESS TM Simplify the microluidic chip design: only 3 inlets and 1 outlet while more than 10 liquids were lown inside the chip As described in this application note, Fluigent ofers high performance solutions, in term of pressure, low-rate and volume control, even for very speciic applications such as rare cells capture and analysis, molecules synthesis and analysis in droplets, chemical mixing reactions, etc. Photograph of captured CTCs identiied with two luorescent markers: Hoechst marker (blue, for DNA) indicating a nucleated cell and cytokeratin (CK, red) indicating an epithelial cell MORE INFORMATION: You can find more information diagrams on the diferent processes (cell capture process automation and cell labelling-bufer selections) on: ACKNOWLEDGEMENT & REFERENCES Institut Curie, MMBM team, Julien Autebert, Benoît Coudert, Stéphanie Descroix, Laurent Malaquin et Jean-Louis Viovy FP7 European Projects: Caminems (and all partners), Diatools (and all partners) ANR project: MiCaD (and all partners) Design, modeling and characterization of microluidic architectures for high fow rate, small footprint microluidic systems, Saias and al., Lab on chip, 2011 Capture and Analysis of Circulating Tumor Cells in Microluidics: From Conception to Clinical Validation, Julien Autebert, Physico-Chimie Curie, UMR168, 2013 (1) Requires FRP (2) On average FOR ANY FURTHER INQUIRY, PLEASE CONTACT US AT: FLUIGENT SA CONTACT@FLUIGENT.COM